Poxviruses encode enzymes and factors needed for transcription and replication of their genomes within the cytoplasm of infected cells. Vaccinia virus, the prototypic member of the poxvirus family, provides a unique system for combining biochemical and genetic approaches for investigating mechanisms of gene regulation, mRNA biosynthesis and DNA synthesis. Studies with vaccinia virus indicated that the genes are divided into three temporal classes - early, intermediate and late. Each gene class has a consensus DNA promoter sequence and corresponding transcription factors that interact with the virus-encoded multisubunit RNA polymerase. The transcription system for early genes is packaged within the infectious virus particle during its assembly, whereas the factors for intermediate and late gene transcription are synthesized successively after infection and localize within cytoplasmic factory areas. Poxviruses also encode enzymes that modify their mRNA by adding a cap structure to the 5' end and a poly(A) tail to the 3' end, which are necessary for efficient translation and stability. The shut down of cellular protein synthesis and the tight regulation of viral protein synthesis are regulated by poxvirus enzymes that cleave the cap structure. Using new generation DNA sequencing, we have made a complete transcription and translation map of the vaccinia virus genome and defined the RNA start sites and the sequences adjacent to the poly(A) tail. These studies have revealed numerous previously unannotated transcripts. In addition, the effects of vaccinia virus infection on host mRNAs have been defined. Vaccinia virus DNA is synthesized within cytoplasmic factories as concatemers that are resolved into unit length genomes and packaged during virus assembly. Studies with conditional lethal mutants indicate that five VACV early proteins are required for DNA replication: namely E9 DNA polymerase, D4 uracil DNA glycosylase, A20 processivity factor, B1 protein kinase and D5 nucleoside triphosphatase (NTPase). The DNA polymerase catalyzes primer- and template-dependent synthesis and possesses 3 to 5 prime exonucleolytic activity. The essential role of D4 in DNA replication is independent of its uracil DNA glycosylase activity, which presumably has a facultative repair function. The A20 and D4 proteins interact and together provide processivity for the DNA polymerase. The B1 kinase was recently shown by others to phosphorylate a cellular DNA-binding protein called BAF and prevent the latter from blocking VACV DNA replication. Potential roles for D5 have come from extensive protein sequence analyses, which indicate that the 90-kDa D5 protein is a member of the helicase superfamily III within the AAA+ class of NTPases, which includes the replicative helicases of some other DNA and RNA viruses. We recently showed that the D5 protein has a second essential function as a DNA primase. In addition, we found that the viral DNA ligase was essential if cellular DNA ligase was inhibited. The two findings of a DNA primase and an essential role for a DNA ligase suggest that poxvirus DNA replication may involve Okazaki fragments. During the past year we demonstrated the presence of a bidirectional replication origin near the ends of the genome. Another recent finding is that a predicted poxvirus FEN1-like nuclease is required for homologous recombination, double-strand break repair and full-size genome formation. During the present year, we provided the first analysis of the viral and host proteins that are associated with replicating viral DNA. During FY 2018, we have undertaken genetic and experimental evolution approaches to further investigate DNA replication and expression. An understanding of the regulation of poxvirus gene expression and DNA replication will help to design vaccines and identify targets for antiviral therapy and will contribute to our understanding of these processes in other viruses and cells.